Our long-term goal is to understand how nitrogen (N) signaling controls plant N-assimilation, growth and development. Identifying key components involved in regulating N-assimilation offers opportunities for improving N-use efficiency in plants or modifying the amino acid content of seeds, which are important issues for health, agriculture and human nutrition. The experiments in this proposal should provide insight into how plants sense and respond to N at the molecular level, specifically through the post transcriptional control of gene expression by microRNAs (miRNAs) and other small RNAs (sRNAs). Identifying the sRNAs involved in regulation of gene networks in response to N-treatment could identify mechanisms which may be altered to optimize N-assimilation via the ectopic expression or misexpression of specific sRNAs. It is now clear that miRNAs are key components of regulatory networks in plants and animals, but their role in mediating the N- response of target genes has not been addressed. We propose to (1) Identify the miRNAs and other sRNAs whose expression is regulated by N-treatments;(2) Identify the gene networks that are controlled by these N- regulated sRNAs;and (3) Understand the functional role of sRNAs in mediating the N-response of target genes in the network. Our experimental approach will be to isolate sRNAs from plants treated transiently with inorganic N sources, sequence the sRNAs using 454 technology, and compare the frequency of transcripts in these samples with the frequency in samples from mock-treated plants. We will incorporate known and novel sRNA:RNA interactions into our existing gene network models to identify subnetworks that are controlled by N- responsive sRNAs. We will use reverse genetic and genomic approaches to evaluate the role of specific sRNAs in the plant response to N-treatments. The combination of bioinformatic and genomic approaches for functional studies in this project performed in collaboration with Dr. Coruzzi's group, will greatly strengthen the research capability of Dr. Gutiirrez research program in Chile and South America. Expertise in these areas is essential for competitive research programs in the post-genomic era, and it is an underdeveloped area of research in Chile and South America. Most of the research will be carried out in Chile at P. Universidad Catslica de Chile in collaboration with Dr. Rodrigo Gutiirrez, as an extension of NIH grant #2R01GM032877- 21 to Dr. Gloria M. Coruzzi. This proposal should provide insight into how plants sense and respond to nitrogen at the molecular level. Identifying key components involved in regulating nitrogen assimilation offers opportunities for improving nitrogen-use efficiency in plants or modifying the amino acid content of seeds, which are important issues for health, agriculture and human nutrition.